This work was supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development (grant HD008913 to C.-H.L.), and the Center for Information Technology (P.G.M., N.P., E.S.M., and M.M.).

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+circuits+of+the+Drosophila+optic+lobe:+the+input+terminals+to+the+medulla.">Synaptic circuits of the Drosophila optic lobe: the input terminals to the medulla.</a> J Comp Neurol. 509 (5), 493-513 (2008).</li><li>Takemura, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+circuits+integrate+neighboring+visual+signals+in+a+Drosophila+motion+detection+pathway.">Cholinergic circuits integrate neighboring visual signals in a Drosophila motion detection pathway.</a> Curr Biol. 21 (24), 2077-2084 (2011).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuriteTracer:+a+novel+ImageJ+plugin+for+automated+quantification+of+neurite+outgrowth.">NeuriteTracer: a novel ImageJ plugin for automated quantification of neurite outgrowth.</a> J Neurosci Methods. 168 (1), 134-139 (2008).</li><li>Scorcioni, R., Polavaram, S., Ascoli, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Measure:+a+web-accessible+tool+for+the+analysis,+comparison+and+search+of+digital+reconstructions+of+neuronal+morphologies.">L-Measure: a web-accessible tool for the analysis, comparison and search of digital reconstructions of neuronal morphologies.</a> Nat Protoc. 3 (5), 866-876 (2008).</li><li>Kaplan, E. 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The authors have nothing to disclose.

Here, we show how to image and analyze dendritic arbors of Drosophila medulla neurons. The first section, dual-view imaging, describes the deconvolution and combination of two image stacks into a high-resolution image stack. The second section, dendrite tracing and registration, describes the tracing and registration of dendrites of medulla neurons to the reference column array. The third section, dendritic analysis, describes the use of custom scripts to analyze dendritic patterns. Together these protocols prov...

During development, neurons elaborate dendrites in complex but stereotyped branched patterns to form synapses with their presynaptic partners. Dendritic branching patterns correlate with neuronal identity and functions. The locations of dendritic arbors determine the type of presynaptic inputs they receive, while the dendritic branching complexity and field sizes govern the input number. Thus, dendritic morphological properties are critical determinants for synaptic connectivity and neuronal computation. In many regions of complex brains, such as the fly optic lobes and the vertebrate retina, synaptic circuits are organized in columns and layers to facilitate information processing1,2. In such a column and layer organization, presynaptic neurons of a distinct modality project axons to terminate at a specific layer (so-called layer-specific targeting) and to form an orderly two-dimensional array (so-called topographic map), while postsynaptic neurons extend dendrites of appropriate sizes in specific layers to receive presynaptic inputs of the correct types and numbers. While axonal targeting to layers and columns has been well studied3,4, much less is known about how dendrites are routed to specific layers and expand appropriately sized receptive fields to form synaptic connections with the correct presynaptic partners5. The difficulty of imaging and quantifying dendritic targeting to layers and columns has hindered the study of dendritic development in columnar and laminated brain structures.
Drosophila medulla neurons are an ideal model for studying dendritic routing and circuit assembly in columns and layers. The fly medulla neuropil is organized as a 3D lattice of 10 layers and approximately 750 columns. Each column is innervated by a set of afferents, including photoreceptors R7/R8 and lamina neurons L1 - L5, whose axonal terminals form topographic maps in a layer-specific fashion6. About 38 types of medulla neurons are present in every medulla column and elaborate dendrites in specific layers and with appropriate field sizes to receive inputs from these afferents7. The synaptic circuits in the medulla have been reconstructed at the electron microscopic level; thus, the synaptic partnerships are well established7,8. Furthermore, genetic tools for labeling various types of medulla neurons are available9,10,11. By examining three types of transmedulla (Tm) neurons (Tm2, Tm9 and Tm20), we have previously identified two cell-type-specific dendritic attributes: (i) Tm neurons project dendrites in either the anterior or posterior direction (planar projection direction), depending on the cell types and (ii) dendrites of medulla neurons terminate in specific medulla layers in a cell-type-specific fashion (layer-specific termination)12. Planar projection direction and layer-specific termination are sufficient to differentiate these three types of Tm neurons, while mutations that disrupt Tm responses to layer and column cues affect distinct aspects of these attributes.
Here, we present a complete workflow for examining the dendritic patterning of Drosophila medulla neurons in columns and layers (Figure 1). First, we show a dual-view imaging method, which uses customized software to combine two confocal image stacks to generate high-quality isotropic images. This method requires only conventional confocal microscopy to generate high-quality images that allow for the reliable tracing of dendritic branches, without resorting to super-resolution microscopy, such as STED (Stimulated Emission Depletion) or structural illumination. Second, we present a method for tracing dendritic arbors and for registering the resulting neurite traces to a reference column array. Third, we show the computational methods for extracting information on the planar projection direction and layer-specific termination of dendrites, as well as for deriving estimates for dendritic branching and termination frequencies. Together, these methods allow for the characterization of dendritic patterns in 3D, the classification of cell types based on dendritic morphologies, and the identification of potential defects in mutants.

<table><tbody><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Huygens Professional</td><td>Scientific Volume Imaging</td><td>version 16.05</td><td>for image deconvolution (https://svi.nl).&amp;nbsp; commercial software</td></tr><tr><td>MIPAV</td><td></td><td>version 7.3.0</td><td>for image recombination and registration (http://mipav.cit.nih.gov/); freeware</td></tr><tr><td>MIPAV plugin: PlugInDrosophila&lt;br/&gt;RetinalRegistration.class</td><td></td><td></td><td>freeware</td></tr><tr><td>MIPAV plugin: PlugInDrosophilaStandard&lt;br/&gt;ColumnRegistration.class</td><td></td><td></td><td>freeware</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>for tracing neurites and assigning reference points for image registration (http://www.bitplane.com); commercial software</td></tr><tr><td>Vaa3D</td><td></td><td></td><td>for visualizing swc files (https://github.com/Vaa3D/release/releases/); freeware</td></tr><tr><td>Matlab</td><td>Mathworks</td><td>R2014b</td><td>for morphometric analysis of dendrites (http://www.mathworks.com); commercial software</td></tr><tr><td>Matlab toolbox: TREES1.14</td><td></td><td>v1.14</td><td>for analyzing dendritic morphometric parameters (http://www.treestoolbox.org/download.html); freeware</td></tr><tr><td>Matlab toolbox: Dendritic_Tree_Toolbox</td><td></td><td>v1.0</td><td>For calculating morphometric parameters (https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration). Freeware</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Sample files&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SWC file definition</td><td></td><td></td><td>http://www.neuronland.org/NLMorphologyConverter/MorphologyFormats/SWC/Spec.html</td></tr><tr><td>The codes and sample files for image combination and registration</td><td></td><td></td><td>https://science.nichd.nih.gov/confluence/display/snc/Data+collections+for+imagines+combination+and+standardize+column+registration</td></tr><tr><td>Reference point example&amp;nbsp;</td><td></td><td></td><td>https://science.nichd.nih.gov/confluence/download/attachments/117216914/points.csv?version=1&amp;amp;modificationDate=&lt;br/&gt;1471880596000&amp;amp;api=v2</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Computer system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MS Windows Windows 7 x64 or Macintosh OS X 10.7 or later</td><td></td><td></td><td>3GHz 64-bit quad-core processor, 16G RAM (minimal)</td></tr><tr><td>Optional: Quadro4000&amp;nbsp; (or above) graphic card</td><td>Nvidia</td><td></td><td>for stereographic visualization of dendrites.</td></tr><tr><td>Optional: NVIDIA 3D vision2</td><td>Nvidia</td><td></td><td>http://www.nvidia.com/object/3d-vision-main.html</td></tr><tr><td>Optional: 120 Hz LCD display for NVIDIA 3D vision2</td><td></td><td></td><td>http://www.nvidia.com/object/3d-vision-system-requirements.html</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24B10 antibody</td><td>The Developmental Studies Hybridoma Bank</td><td>24B10</td><td></td></tr><tr><td>GFP Tag Antibody</td><td>Thermofisher Scientific</td><td>G10362</td><td></td></tr><tr><td>Goat anti-Rabbit (H+L), Alexa Fluor 488</td><td>Thermofisher Scientific</td><td>A11034</td><td></td></tr><tr><td>Goat anti-Mouse (H+L), Alexa Fluor 568</td><td>Thermofisher Scientific</td><td>A21124</td><td></td></tr><tr><td>VECTASHIELD Antifade Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>Mounting&amp;nbsp;Clay&amp;nbsp;</td><td>Fisher</td><td>S04179</td><td></td></tr><tr><td>70% glycerol in 1x PBS</td><td></td><td></td><td></td></tr><tr><td>Cover glasses, high performance, D = 0.17&amp;nbsp;mm</td><td>Zeiss</td><td>474030-9000-000</td><td></td></tr></tbody></table>

analyzing dendritic morphology in columns and layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Using the dual-view imaging procedure presented here, a fly brain containing sparsely labeled Tm20 neurons was imaged in two orthogonal directions. Prior to imaging, the brain was stained with appropriate primary and secondary antibodies for visualizing membrane-tethered GFP and photoreceptor axons. For imaging, the brain was first mounted in the horizontal orientation (Figure 2A,&#160;B). A GFP-labeled Tm20 neuron and the surrounding photoreceptor axons were imaged using...

Watch this Scientific Journal Video about Analyzing Dendritic Morphology in Columns and Layers at JoVE.com

Analyzing Dendritic Morphology in Columns and Layers

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

analyzing-dendritic-morphology-in-columns-and-layers

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9.2K Views.  National Institutes of Health (NIH). Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Video: Analyzing Dendritic Morphology in Columns and Layers

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

Automatic Identification of Dendritic Branches and their Orientation

The structure of neuronal dendritic trees plays a key role in the integration of synaptic inputs in neurons. Therefore, characterization of the morphology of dendrites is essential for a better understanding of neuronal function. However, the complexity of dendritic trees, both when isolated and especially when located within neuronal networks, has not been completely understood. We developed a new computational tool, SOA (Segmentation and Orientation Analysis), which allows automatic measurement of the orientation of dendritic branches from fluorescence images of 2D neuronal cultures. SOA, written in Python, uses segmentation to distinguish dendritic branches from the image background and accumulates a database on the spatial direction of each branch. The database is then used to calculate morphological parameters such as the directional distribution of dendritic branches in a network and the prevalence of parallel dendritic branch growth. The data obtained can be used to detect structural changes in dendrites in response to neuronal activity and to biological and pharmacological stimuli.

Presented is a computational tool that allows simple and direct automatic measurement of orientations of neuronal dendritic branches from 2D fluorescence images.

The structure of neuronal dendritic trees plays a key role in the integration of synaptic inputs in neurons. Therefore, characterization of the morphology ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

Analyzing Dendritic Morphology in Columns and Layers

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Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Automatic Identification of Dendritic Branches and their Orientation

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex